Since the discovery in the mid-eighteenth century that eating oranges or lemons would successfully treat scurvy, vitamin C has been, more or less continuously, used as a dietary supplement. The isolation of naturally occurring L-ascorbic acid (ascorbic acid) was followed shortly by the development of an efficient process for its synthesis. These developments, together with the recognition that its presence in the diet is essential to human health, have lead food manufacturers to attempt to add a source of bio-available vitamin C to, or to enhance the level of naturally occurring vitamin C in, their products. These attempts, however, have been unsuccessful in a significant number of applications, either as a result of the well known autoxidative instability of ascorbic acid or as a result of its reactivity toward other food components.
One example of its reactivity toward other food components, arises when ascorbic acid is present in an aqueous mixture which also contains an anthocyanic pigment. The incompatibility of anthocyanic pigments, i.e., anthocyanins or anthocyanidins, with ascorbic acid is a well established fact in the literature [Sondheimer, E. and Kertesz, Z. I., Food Res., 17, 288 (1952); Starr, M. S. and Francis, F. J., Food Technol. 22, 1293 (1968)]. If both the ascorbic acid and the anthocyanic pigment are present in solution, a chemical reaction occurs which results in the bleaching of the pigments and the destruction of the ascorbic acid. The half-life of anthocyanic pigments at pH 3 and room temperature, for the case of a beverage containing 10 ppm anthocyanidin and 600 ppm ascorbic acid, could be diminished by a factor of as much as 100. This rate of destruction has been found to be increased by increasing temperature and exposure to light.
The mechanism of the ascorbic acid-anthocyanic pigment bleaching reaction has not been conclusively proven, but at least two mechanistic theories appear to have been put forward to explain the phenomenon.
The first mechanistic theory postulates an oxidation-reduction path involving both the ascorbic acid and oxygen. In the publication "Oxygen and Ascorbic Acid Effect on the Relative Stability of Four Anthocyanin Pigments in Cranberry Juice" [Starr, M. S. and Francis, F. J., Food Technology, 22, 1293-95 (1968)], the authors trace the recognition of oxygen involvement in the ascorbic acid bleaching of anthocyanic pigments from a 1943 discovery that increasing air headspace in bottles of strawberry juice containing anthocyanin pigments increased the degradation of the pigment. A 1956 report disclosed that oxygen increased the rate, and extent, of discoloration of the red anthocyanin pigments. The Starr and Francis publication further describes previous research on ascorbic acid bleaching of anthocyanins in the presence of oxygen that supports the theory that a reactive intermediate is formed from an initial oxygen-ascorbic acid reaction (possibly hydrogen peroxide or a free radical species). This intermediate subsquently reacts with the anthocyanic pigment. From their original research reported in this publication, Starr and Francis disclose that anthocyanin bleaching is increased by increasing the concentration of either oxygen or ascorbic acid.
In a more recent publication by Shirkhande and Francis, [Effect of Flavonols on Ascorbic Acid and Anthocyanin Stability in Model Systems, J. Food Sci., 39, 904 (1974)], the authors note both the theory that hydrogen peroxide (formed through a free radical mechanism from oxygen and ascorbic acid) is the reactive intermediate in anthocyanin bleaching and the fact that flavonols retard the autoxidation of ascorbic acid. The authors note also the previously published observation that the simultaneous presence of both flavonols and anthocyanins in ascorbic acid solutions appears to protect the ascorbic acid from oxidation. The results of their original research reported in this publication demonstrate that the addition of the flavonol anti-oxidants quercetin or quercitrin to model systems containing dissolved oxygen, ascorbic acid and four anthocyanins found in cranberries retards both the oxidation of ascorbic acid and the bleaching of the anthocyanins. The authors conclude that these results are consistent with an ascorbic acid-anthocyanin bleaching mechanism which involves a first step of oxygen-ascorbic acid reaction to form either hydrogen peroxide or a free radical species as a reactive intermediate, followed by a second step of reactive intermediate-anthocyanin bleaching.
A second mechanistic theory for ascorbic acid bleaching of anthocyanic pigments has been advanced by Jurd ["Some Advances in the Chemistry of Anthocyanin-Type Plant Pigments", pp. 123-42, in "The Chemistry of Plant Pigments," Co.O. Chichester, Ed., Academic Press, N. Y., 1972, and references therein]. This theory postulates that the bleaching reaction between these two species occurs as a result of a nucleophilic substitution by ascorbic acid at the 4 position of the pigment to yield a colorless, 4-substituted flav-2-ene [ibid, p. 139].
In addition to its tendency to bleach anthocyanin and anthocyanidin pigments, the tendency of ascorbic acid to autoxidize in acidic aqueous solutions in the presence of air has long been known. This autoxidative process is known to be accelerated by copper (II) ions, by the enzyme ascorbic acid oxidase [Dawson, C. R., Ann. N.Y. Acad. Sci., 88, 353 (1960)], and by light [McAlpine, R. D., et al., Can. J. Chem., 51, 1682 (1973)]. Because of this autoxidation phenomenon, and because the autoxidation rate is increased by light and other factors, several research groups have attempted to synthesize derivatives of ascorbic acid which retain some degree of in vivo vitamin C activity but which do not exhibit this autoxidative degradation.
As a result of these efforts, it has been observed that derivatives of ascorbic acid which are substituted at the enolic OH function at the C.sub.2 and/or C.sub.3 positions form a family of vitamin C analogs which are able to resist autoxidation because of the blocking of the ene-diol functional group.
Many of these derivatives, particularly the ascorbyl esters at C.sub.2 and/or C.sub.3, were tested on the scorbutic guinea pig and found to have retained a vitamin C potency equivalent to unsubstituted ascorbic acid. Such retention of vitamin C potency was shown for the 2-O-phosphate [Iami, Y, et al., Jap. J. Pharmacol. 17, 317, (1967)] and the 2-O-benzoate [ibid, 17, 330, (1967)] in the scorbutic guinea pig, and for the 2-O-sulfate in the scorbutic rainbow trout [Halver, J. E., et al., Ann N.Y. Acad. Sci., 258, 81 (1975)] and monkey [Baker, E. M., et al., Ann N.Y Acad. Sci., 258, 72(1975)].
In addition to the aforementioned ascorbyl esters, C.sub.3 and/or C.sub.2 O-alkylated derivatives of ascorbic acid have been shown to exhibit both stability to autoxidation and in vivo vitamin C activity, albeit with the activity at a significantly lower level. For example, the 3-O-methyl ascorbic acid was found to exert an anti-scorbutic effect on the guinea pig of 1/50 of vitamin C when given orally [Gould, B. S., et al., Arch. Biochem., 23, 205 (1949)].
Despite the discovery of such non-autoxidizing vitamin C derivatives, and despite the ever-increasing interest in providing a food containing both bio-available ascorbic acid and anthocyanin or anthocyanidin pigments, the development of such a food has been hindered by the incompatibility between such natural pigments and vitamin C, which has heretofore been perceived to result from either a nucleophilic substitution by ascorbic acid at the 4 position of the anthocyanic pigment or an oxidation-reduction reaction involving ascorbic acid, oxygen and the anthocyanic pigment.